title: Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity.
abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1(-) cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase γ, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.
authors.affiliation: Institut de Recerca, Hospital Universitari de la Vall d'Hebron, Barcelona, Spain; Institut de Recerca l'Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. joanvillarroya@gmail.com
authors.forename: Joan
authors.initials: J
authors.lastname: Villarroya
authors.forename: Mari-Carmen
authors.initials: MC
authors.lastname: Lara
authors.forename: Beatriz
authors.initials: B
authors.lastname: Dorado
authors.forename: Marta
authors.initials: M
authors.lastname: Garrido
authors.forename: Elena
authors.initials: E
authors.lastname: García-Arumí
authors.forename: Anna
authors.initials: A
authors.lastname: Meseguer
authors.forename: Michio
authors.initials: M
authors.lastname: Hirano
authors.forename: Maya R
authors.initials: MR
authors.lastname: Vilà
mesh_terms: D045744:Cell Line, Tumor; D019069:Cell Respiration; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics*; D003576:Electron Transport Complex IV / Q000378:metabolism; D015870:Gene Expression; D020868:Gene Silencing; D018390:Gene Targeting; D006801:Humans; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D028361:Mitochondrial Diseases / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004272:DNA, Mitochondrial; D009711:Nucleotides; D003576:Electron Transport Complex IV; C105339:thymidine kinase 2; D013937:Thymidine Kinase